I am purifying a protein naturally containing His residues (the number of residues are much lesser than the His-tagged ones so binding is a bit weaker). I have previously established a purification method using Ni-sepharose at small scale, which was quite consistent up to the fifth use of the column before doing CIP. Now I'm trying to do the same using Ni-IDA for the reason that I'll be handling a bigger scale and IDA is cheaper. When I tried it at small scale using all the same conditions, the results in the first round is the same as what I had with Ni-sepharose. The problem started at the second and went on until the subsequent rounds as there was an observed shift in the elution pattern of the target protein. It was eluting earlier than expected, together with the impurities at the wash step. Can anyone suggest a way to fix this inconsistency? If possible I want to have a consistent elution profile from the first to at least the fourth round.
To get the same consistent result, after elution wash the column with 1M HCl to remove metal ions and whatever was strongly adsorbed. Load the system with new metal ions, equilibrate and inject. If you still do not see consistency it could be that protein is precipitating blocking adsorption sites. You need to apply then a stronger washing.
Omar Gonzalez-Ortega thanks, but I understand this point and I get the consistent result after I do stripping and re-charging with metal ions. Freshly cleaned and re-charged condition give me that result. What I am interested to know is whether there's a way to get such consistency until at least third time of column use without stripping/re-charging.
Do you have chelating agents in the buffers? Do you see protein proecipitation at the entrance of the column? The adsorption-desorption process will always remove some metal ions. Are you equilibrating the column with both adsorption and desorption buffers before sample injection?
These are my buffers: (A) 50 mM Sodium phosphate pH 7.2, (B) A+500 mM Imidazole. I only equilibrate with the adsorption buffer prior to sample injection. Does it make a huge difference I do equilibration with both?
- Badges
- Science method